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The objective of this pilot study was to evaluate the influence of sampling technique and exposure to different bedding types on the milk microbiome of healthy primiparous cows. Primiparous Holstein cows (n = 20) with no history of clinical mastitis or monthly somatic cell counts >150,000 cells/mL were selected for this study. From each enrolled cow, a composite milk sample was aseptically collected from all 4 mammary quarters (individual quarter somatic cell counts <100,000 cells/mL), 1 individual quarter milk sample was collected using conventional aseptic technique, and 2 individual quarter milk samples were collected directly from the gland cistern using a needle and vacuum tube. All milk samples were cultured using standard milk microbiological techniques and DNA was extracted. Extracted DNA was subjected to PCR and next-generation sequencing for microbiota determination. All samples yielded relatively little total DNA. Amplification of PCR was successful in 45, 40, and 83% of composite, conventional, and cisternal samples, respectively. Bacteria were successfully cultured from 35% of composite milk samples but from none of the quarter milk samples collected using conventional or cisternal sampling techniques. Bacterial DNA sequences were assigned to operational taxonomic units (OTU) based on 97% sequence similarity, and bacterial richness and diversity were determined. Most samples were dominated by low-prevalence OTU and of the 4,051 identified OTU, only 14 were prevalent at more than 1% each. These included bacteria typically recovered from environmental sources. Chao richness was greatest in composite samples and was 636, 347, and 356 for composite, conventional quarter, and cisternal milk samples, respectively. Shannon diversity was similar among sample types and ranged from 3.88 (quarter) to 4.17 (composite). Richness and diversity did not differ by bedding type among cisternal samples, but the power of this pilot study was limited due to small sample size. Despite the small sample size, for milk samples collected from the gland cistern, overall bacterial community composition differed among bedding types. These results demonstrate that sampling technique and bedding type may be associated with the microbiota detected in bovine milk, and we suggest that these variables should be considered in designing and reporting studies about the milk microbiota.

Steroid-induced glaucoma is an iatrogenic condition resulting from the use of glucocorticoids. Glucocorticoids such as dexamethasone (DEX) 1 raise intraocular pressure (IOP) in ϳ40% of patients in the general population, and ϳ6% of these patients will go on to develop glaucoma (1, 2). This condition is similar to primary open angle glaucoma (1-3), and is caused by a restriction in fluid outflow through the trabecular meshwork (TM), resulting in an imbalance between the amount of aqueous humor produced and the amount drained. This imbalance results in a higher IOP.It is thought that an alteration in the cytoskeletal structure or contractile properties of TM cells may result in the disruption of normal fluid flow. In support of this idea, cross-linked actin networks, referred to as CLANs, have been observed with increased frequency in the TM of glaucomatous patients and in glucocorticoid treated anterior segments as well as in TM cells in culture. CLANs are thought to alter the contractility of the TM by holding the cells in a rigid conformation, making the cells unresponsive to the change in pressure and blocking the aqueous humor outflow pathway (1,4,5). Thus, agents such as H7 and the latrunculins A and B, which disrupt the organization of the cytoskeleton, decrease IOP in porcine and monkey cultured anterior segments (6 -9).Control of the actin cytoskeleton is mediated by the Rho family of small GTPases. The Rho effector ROCK has been shown to play a part in TM contractility and modulation of IOP. Inhibition of ROCK using a dominant negative mutant or the inhibitor Y-27632 causes TM cells to "relax" by decreasing actin stress fiber formation and phosphorylation of myosin light chain (MLC) (10, 11). ROCK inhibition also decreases IOP in cultured human and porcine anterior segments (10, 11). In contrast, constitutively active RhoA (RhoA V14) increases From the ‡Departments

ABSTRACT

The objective of this longitudinal cohort study was to describe the milk microbiota of dairy cow mammary glands based on inflammation status before and after the dry period. Individual mammary quarters were assigned to cohorts based on culture results and somatic cell count (SCC) at dryoff and twice in the first 2 weeks post-calving. Mammary glands that were microbiologically negative and had low SCC (< 100,000 cells/mL) at all 3 sampling periods were classified as Healthy (n = 80). Microbiologically negative mammary glands that had SCC ≥150,000 cells/mL at dryoff and the first post-calving sample were classified as Chronic Culture-Negative Inflammation (CHRON; n = 17). Quarters that did not have both culture-negative milk and SCC ≥ 150,000 cells/mL at dryoff but were culture-negative with SCC ≥ 150,000 at both post-calving sampling periods were classified as Culture-Negative New Inflammation (NEWINF; n = 6). Mammary glands with bacterial growth and SCC ≥ 150,000 cells/mL at all 3 periods were classified as Positive (POS; n = 3). Milk samples were collected from all enrolled quarters until 150 days in milk and subjected to microbiota analysis. Milk samples underwent total DNA extraction, a 40-cycle PCR to amplify the V4 region of the bacterial 16S rRNA gene, and next-generation sequencing. Healthy quarters had the lowest rate of PCR and sequencing success (53, 67, 83, and 67% for Healthy, CHRON, NEWINF, and POS, respectively). Chao richness was greatest in milk collected from Healthy quarters and Shannon diversity was greater in milk from Healthy and CHRON quarters than in milk collected from glands in the NEWINF or POS cohorts. Regardless of cohort, season was associated with both richness and diversity, but stage of lactation was not. The most prevalent OTUs included typical gut- and skin-associated bacteria such as those in the phylum Bacteroidetes and the genera Enhydrobacter and Corynebacterium. The increased sequencing success in quarters with worse health outcomes, combined with the lack of bacterial growth in most samples and the high PCR cycle number required for amplification of bacterial DNA, suggests that the milk microbiota of culture-negative, healthy mammary glands is less abundant than that of culture-negative glands with a history of inflammation.

Studies were performed to obtain evidence for glyconeogenesis from pyruvate to the triose phosphates in pancreatic islets. Inability to show this evidence would be consistent with the fact that glyceraldehyde, but not pyruvate, is a potent insulin secretagogue. Synthesis of 14C-labelled glucose from 14C-labelled pyruvate could not be detected. Since this might have been due to lack of sensitivity required to measure 14C-glucose production in such a scarce tissue as islets, cDNA probes were used to estimate the relative expression of genes coding for gluconeogenic enzymes. Islets expressed pyruvate carboxylase mRNA, but even islets from rats which had been starved (a condition which induces phosphoenolpyruvate carboxykinase (PEPCK) in liver, kidney and adipose tissue) showed no PEPCK mRNA. This is consistent with our previous work showing the absence of PEPCK enzyme activity in islets. Therefore, islets can convert pyruvate to oxalacetate, but since they lack PEPCK, neither the beta nor alpha cell can convert oxalacetate to phosphoenolpyruvate and carry out glyconeogenesis. Pyruvate carboxylase mRNA was increased in islets that possessed the capacity for glucose-induced insulin release versus islets that lacked the capacity to respond to glucose, such as islets from fed rats (versus starved rats) and in islets cultured at a high concentration of glucose (versus at low glucose). Pyruvate carboxylase, therefore, must be involved in pyruvate metabolism and not glyconeogenesis in the pancreatic islet.

Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca 2+ -binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca 2+ concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca 2+ concentration. However, binding of Ca 2+ to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca 2+ affinity. The addition of N-terminal ALG-2 peptides, residues 1-22 or residues 7-17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca 2+ binding to EF-3, while Ca 2+ binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition. † This work was supported by NIH Grants EY12768 and EY13705 (A.S.P.), EY08061 (K.P.), and EY06603 and EY14239 (J.W.C.) and the Swiss National Science Foundation 31-65071.01 (J.C.), as well as grants from the Retina Research Foundation, Research to Prevent Blindness Inc. (RPB), the University of Wisconsin Comprehensive Cancer Center, and the E. K. Bishop Foundation. L.S. is a recipient of the Cremer Scholarship. A.S.P. is a Jules and Doris Stein RPB Professor. * To whom correspondence should be addressed. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptApoptosis-linked gene-2 (ALG-2) was first identified in a death-trap assay using a mouse T cell hybridoma model (1). These cells when transfected with an antisense ALG-2 expression vector were resistant to T cell receptor-mediated cell death and were partially protected from other agents that normally initiate programmed cell death such as Fas, staurosporine, actinomycin D, and C 2 -ceramide (1). More recent work suggests that ALG-2 might be involved in the apaf1-independent intrinsic apoptotic pathway activated as a result of endoplasmic reticulum (ER) stress (2).ALG-2 contains five putative EF-hand sequences and is a member of the family of PEF 1 (penta-EF-hand) proteins that includes the calpain small subunit, sorcin, and grancalcin (3). In ALG-2, EF-1 and EF-3 are considered the high-affinity Ca 2+ -binding sites, while one low-affinity site...

Hepatic iron overload is a serious complication of chronic transfusion therapy in patients with sickle cell disease (SCD). No firm consensus has been reached with regard to correlation between hepatic iron content (HIC) and variables including age, number of transfusions, and serum iron makers. Also, the role of HIC in determining hepatic injury is not well established. There is scarcity of data on chronically transfused children with SCD and no other confounding liver pathology. We aimed to further explore relationships between these variables in a cohort of children with SCD on chronic transfusion therapy naive to chelation. Liver biopsies obtained before starting chelation therapy from 27 children with sickle cell anemia receiving chronic transfusion therapy were evaluated for histologic scoring and determination of HIC. Average serum ferritin and iron saturation values were determined for 6 months before biopsy. Duration and total volume of transfusion were obtained from the medical records. All children were negative for human immunodeficiency virus, hepatitis B virus, and hepatitis C virus infections. Mean age at biopsy was 10.95+/-3.34 years. Mean duration and total volume of transfusions were 50.0+/-26.6 months and 17.4+/-9.6 L, respectively. Pearson product-moment bivariate correlation coefficients indicated significant correlations between HIC and histologic iron score, serum ferritin, iron saturation, age, and transfusion volume. After adjusting for transfusion volume, a significant correlation was only seen between HIC and transfusion volume. Mean HIC was 21.8+/-10.4 mg/g dry weight, with fibrosis observed in 10 patients and lobular inflammation in 9. HIC was higher in biopsies with fibrosis (28.2+/-3.8 mg/g) than biopsies without fibrosis (17.6+/-18.3 mg/g; P=0.012). HIC did not differ between biopsies with lobular inflammation (25.5+/-4.0 mg/g) and biopsies without inflammation (19.9+/-2.5 mg/g; P=0.22). These findings show that transfusion volume provides more insight on hepatic iron overload than serum iron markers.